Effects of the Y432S Cancer-Associated Variant on the Reaction Mechanism of Human DNA Polymerase κ

Human polymerases are vital for genetic information management. Their function involves catalyzing the synthesis of DNA strands with unparalleled accuracy, which ensures the fidelity and stability of the human genomic blueprint. Several disease-associated mutations and their functional impact on DNA polymerases have been reported. One particular polymerase, human DNA polymerase kappa (Pol κ), has been reported to be susceptible to several cancer-associated mutations. The Y432S mutation in Pol κ, which is associated with various cancers, is of interest due to its impact on polymerization activity and markedly reduced thermal stability. Here, we have used computational simulations to investigate the functional consequences of the Y432S by means of classical molecular dynamics (MD) and coupled quantum mechanics/molecular mechanics (QM/MM) methods. Our results suggest that Y432S results in structural effects on domains involved in nucleotide addition and ternary complex stabilization while maintaining catalytic competence. Calculation of the minimum energy path associated with the reaction mechanism of wild type (WT) and Y432S Pol κ indicate that while both enzymes are catalytically competent, the cancer mutation results in a slightly endoergic reaction and an increase in the catalytic barrier. Interactions with a third magnesium ion and environmental effects on non-bonded interactions, particularly involving key residues, contribute to the kinetic and thermodynamic distinctions between the WT and mutant during the catalytic reaction. The energetics and electronic findings suggest that active site residues favor the catalytic reaction with dCTP3– over dCTP4–.